The overall objective for this project is to validate, commercialize, and further establish the clinical utility of a comprehensive set of genotyping assays to improve screening and diagnosis of conditions associated with fragile X syndrome (FXS). The relevance of testing for interspersed AGG sequences in CGG repeats has been proposed from population studies of fragile X mental retardation (FMR1) gene expansion. Both the number and sequence context of AGG repeats within the repeat CGG element have been proposed to confer stability to generational expansion. Although AGG interspersions are associated with the risk of triplet repeat expansion, these interspersions previously could not be determined in most female samples. We propose to refine the risk estimates for intermediate and premutation alleles by incorporating AGG interspersion information into the analysis of FMR1 allele transmissions from archived DNA samples using the definitive AGG mapping assays that we have developed. Our assay approach, leverages high throughput PCR and promises improved diagnostic and risk assessments for enabling accurate AGG genotyping, and definitive determination of the number of consecutive CGG repeats for each sample allele. We met or exceeded all proposed aims in the phase I grant. This success has resulted in complementary molecular genetic assays for FMR1 with comprehensive AGG genotyping capabilities. Our academic collaborators are involved in large clinical trials that will examine the genetic factors important for FMR1 repeat expansions, and our technology will be the centerpiece to evaluate the clinical utility of the interspersed AGG mapping for a range of FMR1 disorders. The aims for phase II are: Aim 1: Apply the assay technologies developed in Phase I to retrospective clinical samples to more precisely define the clinical utility of AGG genotyping in CGG repeat expansions. Aim 2: Develop a set of controls and standards for detection of AGG repeats and integrate reagents, controls and QC metrics into a comprehensive workflow that supports the PCR-based detection of AGG genotypes. Aim 3: Develop and test user-friendly software components for mapping AGG positions. Aim 4: Integrate and evaluate comprehensive test features developed in Aims 2 and 3. PUBLIC HEALTH RELEVANCE: We are developing a rapid, and cost-effective molecular test to detect genetic mutation associated with Fragile X Syndrome. Fragile X is one of the most commonly inherited forms of mental retardation and can also cause other disorders and conditions such as ADHD and autism. The diagnostic test is expected to identify people who have a high risk of passing the disorder onto their children.